Compositions and methods for preventing or reducing resistance of insects to insecticides

ABSTRACT

A method for preventing or reducing resistance to a pesticide of a substrate pest, which method comprises the administration to the substrate or the pest of a composition comprising: (a) a rapid-release formulation of an inhibitor of a factor causing or contributing to the resistance of the pest to the pesticide; and, substantially simultaneously, (b) a non-rapid release formulation of the pesticide. The invention also provides compositions suitable for use in such a method.

The present invention relates to a method for preventing or reducingresistance of a pest to a pesticide and to formulations for use in sucha method. In particular, the invention relates to insecticide resistanceand to insecticidal compositions.

There are several definitions of insecticide resistance, oftenreflecting the interest of the scientist attempting the definition,rather than the phenomenon itself. The World Health Organisation hasdefined resistance as “the development of an ability in a strain ofinsects to tolerate doses of toxicant that would prove lethal to themajority of individuals in a normal population of the same species”.Pesticide resistance is therefore to be similarly construed, althoughthe main pests addressed herein are insects.

Insecticide resistance has become progressively more widespread sincefirst being scientifically recorded in 1914. Over 500 insect and mitespecies now show tolerance to pesticides, and pesticide resistance hasbecome a serious threat to the future success of pest control usingchemicals.

There are three major mechanisms by which resistance can occur: reducedpenetration of the pest by the pesticide; metabolism of the insecticide(resulting in detoxification); and target-site insensitivity. Theseresistance mechanisms may exist individually in an insect, but are oftenfound in combination where the overall resistance offered issubstantially higher; this situation is referred to as ‘multi-factorialresistance’.

Many insects possess detoxification systems, which evolved originally toprotect the insect from natural toxins in the environment. Metabolism ofthe insecticide may occur before it reaches its target-site when itcomes into contact with those detoxifying enzymes that render it eitherless toxic or more easily excreted, or both. The most important enzymesystems involved in insecticide resistance include the groups a) mixedfunction oxidases, b) glutathione S-transferases and c) esterases.Resistance resulting from enhanced activity of one or more of theseenzyme groups has been found in several insect species.

Insect detoxifying enzyme systems can be studied either in vivo byconventional bioassays, or in vitro by biochemical assays. Inconventional bioassays, there is widespread employment of synergistssuch as DEF (S,S,S-tributyl phosphorothioate) and TPP (O,O,O-triphenylphosphate). These are compounds that significantly enhance the toxicityof an insecticide, although they may be virtually non-toxic when usedalone. Insecticide synergists act by inhibiting metabolic enzymes.Mortality differences in a bioassay, using a pesticide in the presenceor absence of a synergist, should indicate whether a putative metabolicenzyme is involved in resistance. However, caution should be taken whenusing synergists; very often the chemical is not completely specific tothe enzyme being examined, and it may be difficult to assess itspossible effect upon other biological systems.

Esterases are enzymes that catalyse the hydrolysis of an ester bond.Organophosphate, carbamate and most pyrethroid insecticides containester bonds and in some instances are sensitive to hydrolysis byesterases.

Esterases can act by either sequestering toxins to the insect or byhydrolysing the toxins. Therefore resistance to insecticides can resultfrom either quantitative or qualitative changes in carboxylesterases, ora combination of the two. Qualitative changes could confer to the enzymethe ability to hydrolyse insecticidal esters at a significant rate, butmay or may not affect the activity of the esterase towards the modelsubstrates. Without a qualitative change, resistance can still occur byquantitative changes resulting from a process of gene amplification.This leads to the production of a greater amount of the same esterase,which sequesters the insecticide, resulting in resistance. Occasionally,the esterase may be both altered and amplified.

Piperonyl butoxide (PB or PBO) has been used extensively as a ‘tankmix’, both as an excipient due to its detergent/surfactant properties,and because of the wealth of literature describing its ability toinhibit oxidative metabolic enzymes (mixed function oxidases). We haveshown that certain non-specific esterases involved in pesticideresistance are partially inhibited by micromolar concentrations ofpiperonyl butoxide (IUPAC, London 1998).

In Australian Helicoverpa armigera (Hübner), up to 70% of the activityof pyrethroid-resistance related esterases was inhibited by 10⁻⁵ Mpiperonyl butoxide, both in homogenates of resistant insects and in apartially-purified esterase extract . (Gunning et al in PiperonylButoxide, pp 215-25, Academic Press (1998)).

Studies were also performed on esterases from the cotton aphid, Aphisgossypii (Glover) and the peach-potato aphid, Myzus persicae (Sulzer).Piperonyl butoxide was capable of inhibiting esterase activity from A.gossypii, but only when present at nominal concentrations of 10⁻⁴ M orgreater. Total esterase activity was typically reduced by 50% in 30minutes. This effect is not simply a consequence of a physico-chemicaleffect involving the substrate, since esterases present in M. persicaedirectly implicated in insecticide resistance were not inhibited whenincubated with mM concentrations of piperonyl butoxide for 40 minutes.

Gunning et al (1998) therefore proposed the use of a synergist oresterase inhibitor such as PBO simultaneously in a tank mix with aninsecticide such as pyrethroid to improve efficacy of the insecticide inthe field. Furthermore, data obtained by Gunning et al (Pest Biochem &Physiol 63 50-62 (1999)) reveal significant pyrethroid synergism byorgano-phosphates; in earlier studies, workers in the field did notobserve this effect, doubtless because the pre-treatment period used insuch studies (profenofos and DEF) never exceeded 30 minutes, which istoo short a period for such an effect to become evident.

Thus, in some cases where resistance is conferred by esteratic enzymes,PBO or similarly acting analogue of PBO, such as a UV stable variantthereof, could be added to inhibit the esterases for a period of timeprior to the addition of a conventional insecticide. This would normallynecessitate a second insecticide application, ie a pre-treatment with ametabolic enzyme inhibitor prior to insecticide spray, which is not aneconomic proposition compared to a single application e.g. of the tankmix.

The present invention overcomes the problem of multiple application byproposing that, if an insecticide were microencapsulated or otherwiseadministered in a non-immediate release formulation and the PBO or otheresterase inhibitor not so, then a single application would suffice. ThePBO would immediately begin to act on the esterases and, after a givenperiod, the micro-encapsulation would break down and release theconventional insecticide. By this time, the resistance-associatedenzymes would be inhibited, and thus the resistance mechanism overcome.

Various formulations involving both a synergist, such as PBO, and aninsecticide, such as a pyrethroid are known.

European patent specification no. 238 184 relates to the use of amicroencapsulated pesticide and a non-micro-encapsulated pesticide,wherein the two pesticides are preferably the same, eg permethrin.European patent specification no. 427 991 discloses a mixture of amicroencapsulated organophosphorous and/or carbamate pesticide with aflowable phase comprising a pyrethroid pesticide. Both of thesespecifications suggest the use of the formulation for kill-knock downcombined action, as does the German patent specification no. 2411 373,which discloses a partly micro encapsulated formulation of a pyrethroid,optionally containing a synergist. The entire text of all three of theseearlier patent applications is hereby incorporated by reference.However, none of these formulations relates to one suitable for thepurposes of this invention, namely to reduce or prevent pesticideresistance by enabling an esterase inhibitor to come into contact withthe pest first, followed by the pesticide, in a single application.

Accordingly, the present invention provides a method for preventing orreducing resistance to a pesticide by a pest, which method comprises theadministration to the crop, other substrate or the pest of a compositioncomprising:

-   -   (a) a rapid-release formulation of an inhibitor of a factor        causing or contributing to the resistance of the pest to the        pesticide; and, substantially simultaneously,    -   (b) a non-rapid-release formulation of the pesticide.

Furthermore, the present invention provides a composition, suitable foruse in such a method, which composition comprises:

-   -   (a) a rapid-release formulation of an inhibitor of a factor        causing or contributing to the resistance of the pest to the        pesticide; and, substantially simultaneously,    -   (b) a non-rapid-release formulation of the pesticide.

Preferably, the rapid release formulation and the sustained releaseformulation are comprised in the composition in physical admixture.However, the formulations (a) and (b) may be administered separately. By‘substantially simultaneously’ herein is meant that the formulations arebrought into contact with the substrate and/or the pest at about thesame time, avoiding the need to revisit the site of the substrate and/orpest in order to apply the second of the two formulations. Bothformulations would thereby come into contact with the substrate and/orthe pest within the order of seconds, preferably within 10 seconds andmore preferably, within one or two seconds, of each other rather than inthe order-of minutes or longer. Preferably, the formulations (a) and (b)are administered simultaneously.

The rapid release formulation is suitably any standard pesticideformulation known to those skilled in the art or yet to be discoveredand suitable for the purpose. Such formulations include, for example,wettable powders, granulates, emulsifiable concentrates and ultra-lowvolume formulations to which water can be added to form an emulsion, asuspension and the like. Preferably, the rapid release formulation,comprising PBO or other metabolic enzyme inhibitor, is in the form of anemulsifiable concentrate. It will be appreciated that the preferredenzyme inhibitor, or combination of enzyme inhibitors, will be selectedon the basis of which pesticide compound or compounds are being employedagainst a specific pest.

The non-rapid release formulation is suitably any non-immediate releaseformulation known in the art or yet to be discovered, such as sustained,controlled or slow release formulations suitable for the purpose.Preferably, the non-rapid release formulation is one that prevents aneffective dose of the pesticide from being released or coming intoeffective contact with the pest or its target in the pest until theesterase inhibitor, or inhibitor of another factor causing orcontributing to pesticide resistance, has at least begun its inhibitingeffect on its target in the pest. Suitably, the non-rapid releaseformulation prevents release of the pesticide or contact thereof withthe pest or the substrate for at least 30 after application of thecomposition. Such formulations include, for example, the pesticideencapsulated in a degradable capsule and preferably comprisemicro-encapsulation technology. One such example of a surface sprayencapsulating a pyrethroid insecticide is Karate Zeon [trademark](lambda-cyhalothrin). The optimal time delay for release of thepesticide will be determined by a number of factors and will requireexperimentation to determine the time/response profile of theinhibitor(s) selected. A non-release formulation which corresponds withthis profile will then be selected/developed.

Suitable micro-encapsulation formulations include those analogous tothose described in the aforementioned European and German patentspecifications but adapted so as to microencapsulate the insecticide (egpyrethroid) and not the metabolic enzyme inhibitor (eg esteraseinhibitor, eg PBO).

The pesticide itself is suitably any that is capable of acting as suchand to which resistance has been identified amongst the, or some of the,pest(s) against which it is otherwise active. Examples of suitablepesticides that may comprise the active ingredients of component (b) ofthe composition therefore include pyrethroids, organo-phosphates andcarbamates. Preferably, the pesticide is a pyrethroid, such asfenvalerate, s-fenvalerate, cypermethrin (both alpha and zeta forms),bifenthrin, deltamethin and beta-cyfluthrin. It will be appreciated thatnew pesticides and new classes of pesticides are discovered from time totime, and that resistance to pesticides can develop over time. It isintended that the principles of this invention, and the inventiveconcepts therein, can be applied to a wide range of pesticides, bothknown and those yet to be discovered, as and when resistance isidentified.

Component (b) therefore preferably comprises an amount equivalent to astandard dosage of the pesticide. For example, in the case ofbeta-cyfluthrin for pesticidal activity against Helicoverpa, a typicaldose comprises 8 g/L of an ultra-low volume or 25 g/L of an emulsifiableformulation; and for alpha-cypermethrin a typical dose comprises 16 g/Lof an ultra-low volume or 100 g/L of an emulsifiable formulation.

The inhibitor is suitably any that is capable of preventing or reducingresistance of the pest(s) to the pesticide. Suitable inhibitorstherefore include esterase inhibitors, microsomal oxidase inhibitors andglutathione S-transferase inhibitors. Preferably, the inhibitor is anesterase inhibitor, such as PBO, ethion, profenofos and dimethoate.

Component (a) preferably comprises an amount of the inhibitor sufficientto prevent or reduce the resistance of the pest(s) to the pesticide andwill depend on pest size (eg a white fly needs a lot less inhibitor thanan H. armigera grub), degree of ester-mediated resistance etc, but isdeterminable by those skilled in the art.

The pest(s) against which the composition of the invention is/aredirected can be any which are known to offer at least some resistance toa pesticide and which it is considered necessary to disable and/or kill.Examples include those that attack or damage or otherwise reduce thecommercial or other value of a substrate, such as crops, particularlyarable crops, such as food and material crops including cotton. Otherpests include those that are a nuisance to or an adversary of otherliving organisms, including mammals, such as humans.

Accordingly, the pest(s) may include one or more of Helicoverpaarmigera, Helicoverpa punctigera, Heliothis virescens, Aphis gossypii,Myzus persicae, P. includens, W. cervinata, Bemisia tabaci and mosquitospecies.

By way of example, the cotton bollworm Helicoverpa armigera andB-biotype B. tabaci (Poinsettia or silverleaf whitefly) are major croppests worldwide. Extreme insecticide resistance exacerbates the peststatus of these insects. Pyrethroid and Other resistances in AustralianH. armigera and B-biotype B. tabaci are caused by an over production ofesterase isoenzymes which sequester and metabolise insecticides.

For administration to the substrate, any method known in the art forapplication of a pesticide or the like to a substrate may be used andmay depend upon factors such as the particular substrate (eg crop),target pest stage of the crop and the like. Examples of such methodsinclude spraying by ground or aerial application. For administration tocrops, particularly over vast areas such as the Australian cottonfields, it is preferred to spray a composition comprising a suspensionor emulsion of the components (a) and (b) in water, optionally alsocomprising a surfactant or other excipients, (although PBO itself canact as a surfactant) or an ultra-low volume (omitting the water)composition, supplied in a tank, such as one adapted to be transportedby aircraft or, for example as in the case of whitefly sprays, by groundrig (such as tractor, tank or boom spray).

The rate of administration of the compositions according to theinvention will accord with known or approved (registered) rates of theactive ingredients of each of the formulations (a) and (b). For example,for H. armigera, the registered rate in Australia for PBO is in therange of from 250-360 g a.i./ha and for a pyrethroid rates is in therange of from about 12-80 g a.i./ha.

The present invention therefore further provides:

-   -   (a) the use of a composition according to the invention in the        treatment or prevention of pesticide resistance;    -   (b) the use of a composition according to the invention in the        treatment or prevention of damage to or destruction of a        substrate by a pest;    -   (c) the use of a composition according to the invention in pest        control; and    -   (d) a method for preparing a composition according to the        invention, which method comprises bringing the components (a)        and (b) into physical admixture.

The present invention will now be illustrated by the following Examples.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described, by way of example only, withreference to the following Figures wherein:

FIG. 1 shows percentage H. armigera esterase activity (expressed as % ofcontrol, ±standard deviation) remaining at fixed periods followingtopical application of 1 μl of 1% PBO;

FIG. 2 shows percentage B-type B. tabaci (Australian) esterase activity(expressed as % of control, ±standard deviation) remaining at fixedperiods following exposure to 0.1% PBO;

FIGS. 3 & 4 show comparison of percentage esterase inhibition by H.armigera larvae using PBO pre-treatment, fenvalerate andzeta-cypermethrin;

FIG. 5 shows rate of onset of symptoms of pyrethroid poisoning (indaylight and night) in 3^(rd) instar pyrethroid susceptible H. armigeralarvae. Larvae were treated, by topical application, with adiscriminating dose of lambdacyhalothrin using either Karate or KarateZeon;

FIG. 6 shows toxicity, using a topical application bioassay, of KarateEC and Karate Zeon and mixtures of piperonyl butoxide (1%) and Karate ECand Karate Zeon to pyrethroid susceptible and resistant (80 foldresistant to lambdacyhalothrin) 3^(rd) instar H. armigera. Insecticideswere applied to H. armigera at night (under red light) and bioassayswere left overnight in darkness;

FIG. 7 shows toxicity, using a leaf dip bioassay, of Karate EC andKarate Zeon and mixtures of piperonyl butoxide (1%) and Karate EC andKarate Zeon to adult pyrethroid susceptible native B. tabaci andresistant (2000 fold resistant to lambdacyhalothrin) B-biotype B.tabaci. Insecticides were applied to B. tabaci at night (under redlight) and bioassays were left overnight in darkness;

FIG. 8 shows field control on cotton of pyrethroid resistant, secondinstar H. armigera (80 fold to lambdacyhalothrin), using registeredrates of delayed release Karate Zeon and immediate release Karate EC,and mixtures of Karate Zeon and Karate EC with PBO. Error bars representstandard deviations. (Rates of insecticide applied were: PBO 320 ga.i./ha, and lambdacyhalothrin 15 g a.i./ha);

FIG. 9 shows field control on cotton of pyrethroid resistant, B-biotypeB. tabaci adults, using sprays of registered rates of delayed releaseKarate Zeon and immediate release Karate EC, and mixtures of Karate Zeonand Karate EC with piperonyl butoxide). Sprays were applied to cottonunder heavily overcast light conditions. Error bars represent standarddeviations. (Rates of insecticide applied were: PBO 320 g a.i./ha, andlambdacyhalothrin 15 g a.i./ha).

EXAMPLES General Methods & Materials

Esterase activity was determined by measuring the rate of hydrolysis ofthe model substrate, 1-naphthyl acetate, by carboxylesterases present inorgano-phosphate resistant insects such as H. armigera, or thehydrolysis of 1-naphthyl buturate for B. tabacii. Such hydrolysis willresult in a characteristic yellow/brown colour after complex with FBRR(fast blue RR salt) with absorbance at 450 nm, which is measured todetermine the reaction rate. FBRR (0.6% of final solution), wasdissolved in pH6.0, 0.2M phosphate buffer (0.5L), then 1.86% 1-naphthylacetate or 1-naphthyl butyrate was added.

Kinetic assays were performed using a Bio-Rad 3550 micro plate reader(Bio-Rad Laboratories, UK using Kinetic Collector 2.0 software run on aMackintosh SE micro-computer), taking absorbance readings at 450 nmautomatically at 14-second intervals for 10 minutes. The rate wascalculated by the online computer as the slope of the fitted regressionline, using an absorbance limit of 2.0; readings are given in milli-OD(unit of optical density).

Insecticides used were technical grade: fenvalerate (98%, Shell)(R-(R*,S*))-4-chloro-α(1-methylethyl)benzene acetic acid, cyano(3-phenoxyphenyl) methyl ester); cypermethrin((R,S)-alpha-cyano-3-phenoxybenzyl-(1RS)-cis,trans-3-(2,2-dichloro-vinyl)-S,S-dimethylcyclopropane-carboxylate);and zeta-cypermethrin((S)-cyano(3-phenoxyphenyl)-methyl(±)-cis-trans-3-(2,2,-dichloroethenyl)-2,2-dimethylcyclo-propanecarboxylate)(85%, FMC). The insecticide synergist, piperonyl butoxide (96% pure,technical grade), and an 800 g/l emulsifiable concentrate formulation ofthis chemical (PBEC80) were supplied by Endura Spa, Bologna, Italy.

Example 1 PBO Inhibits H. armigera Esterases

Kinetic assays confirmed that esterase activity was inhibited by theinsecticide synergist, PBO, over a 24-hour period (FIG. 1), providingevidence that PBO inhibits H. armigera esterases. In addition, kineticassays illustrate that esterase inhibition by PBO does not occurimmediately after dosage, but occurs with maximum enzyme inhibition from3 to 4 hours after (70 to 72% esterase activity inhibition). Generally,esterases begin to gradually recover until full esterase activity ispresent at 24 hrs. However, it should be noted that percentage esteraseof control remains at less than 50% between 2 and 11 hrs.

Example 2 PBO Inhibits B-Type B. tabaci Esterases

Kinetic assays also showed that PBO inhibits B-type B. tabaci esterasesover a 26-hour period. After an initial rapid inhibition of esterases(by 1 hour), there is a gradual decrease to maximum esterase inhibition(36% of control at 11 hours), prior to a gradual recovery in esteraseactivity with full esterase activity witnessed, 30 hours after initialPBO exposure (FIG. 2). Percentage activity of the control remains atless than 50% between 7.5 and 17 hours and, overall, esterases suffersome degree of inhibition between 1 and 26 hours.

Example 3 PBO Increases Pyrethroid Mortality

Synergism studies confirmed that PBO increases pyrethroid mortality(FIGS. 3 & 4). These involved a comparison of esterase inhibition(expressed as % of control, ±standard deviation) incurred by H. armigeralarvae over time following topical application of 1 μl PBO (1%), and theeffect on mortality of pyrethroid-resistant larvae when exposed toincreasing PBO (1 μl 1,1% PBO/larva) pre-treatment intervals beforefenvalerate (1 μl, 0.125% fenvalerate/larva, FIG. 3) andzeta-cypermethrin (1 μl, 0.01% zeta-cypermethrin/larva, FIG. 4)exposure. Effects were more pronounced with zeta-cypermethrin. There isa highly significant (p<0.01) increase in mortality, until a plateau(100% mortality) is reached (4-5 hrs for fenvalerate, and 4-10 hrs forzeta-cypermehrin); thereafter, the synergistic effects decline. Thistrend corresponds with earlier findings, where esterases are inhibitedto a high degree (more than 50% reduction in esterase activity) between4 and 18 hours.

Example 4 Composition

The following ingredients may be mixed together in water to form acomposition suitable for application from ground or air at standardrates for the pyrethroid:

Formulation (a): 800 g/L PBO (PBO EC formulation)

Formulation (b): 250 g/L Lambda-cyhalothrin (in the form of Karate-Zeon(trademark)); (Karate-Zeon is 250 g/L a.i.)

Example 5 Laboratory Studies with H. armigera Introduction

Laboratory bioassays on pyrethroid resistant and susceptible H. armigerawere conducted in darkness to delay the release of pyrethroid frommicroencapsulation using Karate Zeon®.

Karate Zeon® is a microencapsulated formulation of the pyrethroidlambdacyhalothrin and is the only encapsulated insecticide on theAustralian field crop market. Developed to increase operator safety,this formulation provides a delayed lambdacyhalothrin release (insunlight after mixing with water), of approximately 30 minutes, Releaseof the microcapsule contents is partially triggered by sunlight. A30-minute delay in pyrethroid release is, however, insufficient to allowthe maximal synergist action needed for control of resistant insects.Nonetheless, pyrethroid release in Karate Zeon®, can be delayed beyond30 min by reducing light conditions.

To demonstrate proof of the concept of control of insecticideresistance, using a simultaneous application of a synergist and adelayed release insecticide, we used Karate Zeon® and artificiallydelayed pyrethroid release from encapsulation by using the insecticidein darkness. However, the technology required to prepare delayed releaseinsecticide formulations with a longer time delay to release is known tothose skilled in the art. Thus, microencapsulation techniques may beapplied and adapted to give the desired time delay with a specificinsecticide.

General Methods

H. armigera populations used were: pyrethroid susceptible strain and apyrethroid selected, resistant strain (approximately 80 fold resistantto lambdacyhalothrin). Third instar pyrethroid resistant and susceptibleH. armigera larvae were treated with the insecticide synergist piperonylbutoxide (PBO) and two formulations of lambdacyhalthrin Insecticidesused were: piperonyl butoxide (800 g/L ai), non-encapsulated Karate EC®(50 g/L ai), microencapsulated Karate Zeon® (250 g/L ai). Insecticideswere serially diluted in water. Insecticides were applied topically tolarvae, using a standard, Helicoverpa bioassay procedure (Gunning et al,1984). Experiments were conducted at 25° C. Mortality was assesses after24 h. Control groups were treated with water or PBO and there was nocontrol mortality. Full dosage mortality curves were plotted. Data wereanalysed by probit analysis.

Proof of Delay of Pyrethroid Release in Darkness

Pyrethroids are neuro-toxins affecting the insect peripheral nervoussystem and symptoms of poisoning in H. armigera are well known Gunning,R. V. (Bioassay for detecting pyrethroid nerve insensitivity inAustralian Helicoverpa armigera, Journal of Economic Entomology,89:816-819, 1996). Time of delay of pyrethroid release was estimated(using treatments of Karate EC and Zeon Karate), by recording time tofirst onset of pyrethroid poisoning symptoms in pyrethroid susceptibleH. armigera. Larvae were treated with a dose known to kill 100% ofsusceptible H. armigera larvae, both in strong daylight and in darkness.Three replicates of 30 insects were dosed for each treatment. Nightobservations of larvae were made under red light (insects cannot see redlight).

Results (FIG. 5) show that poisoning symptoms developed in H. armigeratreated with non-encapsulated Karate EC in approximately 30 minutes,both in daylight and darkness. Using encapsulated Karate Zeon, poisoningsymptoms developed in approximately one hour in daylight, while darkconditions delayed the onset of poisoning symptoms until 4.5 h. Thus,use of Karate Zeon in darkness delayed pyrethroid release from itsmicroencapsulation by approximately 3.5h.

Night Bioassays with Pyrethroid Resistant H. armigera

Karate EC and Karate Zeon were serially diluted in water to form a rangeof concentrations for bioassay (0.005-10 μg lambdacyhalothrin/A. Groupsof pyrethroid resistant or susceptible larvae (n=30) received thefollowing insecticide treatments under red light and were held indarkness:

Susceptible Strain

Karate EC, Karate Zeon

Resistant Strain

Karate, Karate EC+PBO, Karate Zeon. Karate Zeon+PBO. Each insectreceived a dose of 10 μg of PBO

TABLE 1 Probit analysis of response of pyrethroid resistant andsusceptible H. armigera to night bioassays of formulations oflambdacyhalothrin and piperonyl butoxide LD₅₀ Fiducial ResistanceTreatment Slope (μg/larva) limits factor Sus. Karate EC 2.1 0.0130.008-0.020 — Sus. Karate Zeon 2.2 0.014 0.008-0.023 — R. Karate EC 1.30.60 0.45-0.87 46 R. Karate Zeon 1.3 0.60 0.45-0.82 46 R. Karate EC +PBO 1.3 0.33 0.25-0.45 25 R. Karate Zeon + PBO 2.1 0.013 0.008-0.02  1

Bioassay results are shown in Table 1 and FIG. 6. The toxicities ofKarate EC and Karate Zeon to susceptible larvae were not significantlydifferent. Toxicities of Karate EC and Karate Zeon to resistant H.armigera were also indistinguishable (46 fold resistance factor). PBOand delayed release Karate Zeon completely overcame resistance (RF=1),while a PBO and Karate EC reduced the level of resistance to 25 fold.

Conclusions

H. armigera treated with PBO and delayed release Karate Zeon becameeffectively susceptible to lambdacyhalothrin with complete suppressionof resistance. Night use of Karate EC+PBO incompletely suppressedresistance, further emphasising that, in order to control resistantinsects, a delay between PBO application and pyrethroid release isnecessary for optimal esterase inhibition by PBO.

Example 6 Laboratory Studies with B-biotype bemisla tabaci Introduction

Laboratory bioassays on pyrethroid resistant and susceptible B-biotypeB. tabaci were conducted in darkness to delay the release of pyrethroidfrom microencapsulation using Karate Zeon®.

General Methods

Pyrethroid susceptible (Northern Australian native B. tabaci) andresistant B-biotype B. tabaci adults (˜2000 resistant fold tolambdacyhalothrin) were treated with formulated insecticide synergistpiperonyl butoxide and two formulations of lambdacyhalothrin(non-encapsulated Karate EC® and microencapsulated Karate Zeon®).Insecticides used were piperonyl butoxide 800 g/L EC, Karate EC (50 g/LEC) and Karate Zeon (250 g/L).

Formulated lambdacyhalothrin was serially diluted in water to form anumber of test concentrations (0.1-10000 ppm lambdacyhalothrin). Astandard leaf dip bioassay technique for adult whiteflies was used(Cahill 1995). Cotton leaf discs were dipped in lambdacyhalothrinconcentrations in a mixture containing 1% PBO. The leaves were dried andplaced on an agar bed in petri dishes. Adult whiteflies were added andthe peri dishes sealed. Bioassays were conducted at night at 25° C.Water dipped and PBO controls were performed. Mortality was assessed andcorrected for control mortality (which did not exceed 5%) Full doseresponse curves were plotted and data analysed by probit analysis.

Results

Dosage mortality data are shown in Table 2 and FIG. 7. Data fromsusceptible B. tabaci show no differences between the toxicity of KarateEC and Karate Zeon. There were also no differences between the toxicityof Karate EC and Karate Zeon to resistant B. tabaci (RF˜140). Pyrethroidresistant B. tabaci treated with PBO and delayed release Karate Zeonwere indistinguishable from susceptible strains in response tolambdacyhalothrin (RF=1), while treatment with PBO and Karate EC reducedresistance to lambdacyhalothrin somewhat (RF=52).

TABLE 2 Probit analysis of the response of pyrethroid resistant andsusceptible H. armigera to night bioassays of formulations oflambdacyhalothrin and piperonyl butoxide LD₅₀ Fidicial ResistanceTreatment slope (ppm) limits factor Sus Karate EC 3.0 0.60 0.50-0.73 —Sus Karate Zeon 3.0 0.61 0.48-0.73 — R. Karate EC 0.90 87.3  59-129 146R. Karate Zeon 0.86 81  54-122 135 R. Karate EC + PBO 0.89 31.1 21-46 52R. Karate Zeon + PBO 2.96 0.58 0.48-0.70 1

Conclusions

B. tabaci treated with PBO and delayed release Karate Zeon becameeffectively susceptible to lambdacyhalothrin with complete suppressionof resistance. Night use of Karate EC+PBO incompletely suppressedresistance, further emphasising that, in order to control resistantinsects, a delay between PBO application pyrethroid release is necessaryfor optimal esterase inhibition by PBO.

Example 7 Field Studies with a Synergist and Delayed Release Pyrethroidon Cotton against h. armigera—Night Sprays Introduction

The H. armigera laboratory studies described above were followed up witha small-scale replicated field trial on conventional cotton at theAustralian Cotton Research Institute at Narrabri, NSW, February 2003.

Trial Method

In the lack of H. armigera pressure on cotton, pyrethroid resistantsecond instar H. armigera larvae, which were the progeny a field strainoriginating from Queensland, were placed on cotton plant. The strain was20 fold resistant to lambdacyhalothrin.

Insecticides used were piperonyl butoxide 800 g/L a.i EC, Karate EC (50g/L EC a.i) and Karate Zeon (250 g/L a.i). Insecticides were mixed withwater. Insecticides were sprayed at registered rates on cotton of PBO320 g a.i. /ha, and lambdacyhalthrin 15 g a.i/ha, using a calibratedhand held boom spray Treatments were: an untreated control, PBO control,Karate EC, Karate Zeon and Karate EC and Karate Zeon mixed with PBO.

The trial was conducted on replicated, 1 row×2m plots. Each plotcontained from 13-17 mature cotton plants. There was an unsprayed bufferof two rows between each plot. Just prior to sunset, ten second instarH. armigera larvae were placed onto the terminals of each plant in thetest plots and the plots sprayed. H. armigera numbers per plant wereassessed one day after treatment. Temperature ranged from 24-26° C. Themean percentage mortality and standard deviation were calculated foreach treatment. There was no mortality in the control.

Results

Results (FIG. 8) show that lambdacyhalothrin controlled ˜40% of H.armigera irrespective of formulation, treatment with a PBO+Karate EC mixdid not give significantly increase mortality. However, PBO mixed withKarate Zeon gave greater than 90% mortality of resistant insects,indicating almost complete suppression of resistance. These consistentwith the results of the laboratory bioassays.

Conclusions

These field data demonstrate that the synergist piperonyl butoxide, whenapplied simultaneously with a delayed release pyrethroid, providedeffective field control of pyrethroid resistant H. armigera. Delayedrelease of pyrethroid allowed time for the synergist to inhibitresistance-associated esterases, providing 100% greater control ofpyrethroid resistant H. armigera than PBO mixed with a non-encapsulatedKarate EC.

Example 8 Field Studies with a Synergist and Delayed Release Pyrethroidon Cotton Against B-Biotype b. tabaci—Reduced Light ConditionsIntroduction

The B. tabaci laboratory studies described above, were followed up witha small-scale, replicated field trial on conventional, commercial cottonat Emerald, Qld, February 2003. Since rain and high wind prevented anynight spraying, insecticides were applied in the morning and the trialconducted under greatly reduced light (heavily overcast, low cloud andrain showers), compared to normal daylight conditions.

Trial Method

The trial was conducted on mature cotton with low B. tabaci pressure (˜2whitflies/terminal). Whiteflies were approximately 100 fold resistant tolambdacyhalothrin

Insecticides used were piperonyl butoxide 800 g/L a.i EC, Karate EC (50g/L EC a.i) and Karate Zeon (250 g/L a.i). Insecticides were mixed withwater and sprayed at registered rates on cotton (PBO 320 g a.i./ha, andlambdacyhalthrin 15 g a.i/ha) using a calibrated hand held boom spray.Treatments were: an unsprayed control, PBO control, and both Karate ECand Karate Zeon mixed with PBO.

The trial was conducted on replicated, 1 row×10 m plots. There was anunsprayed buffer of two rows between each plot. Whitefly numbers wereassessed, by counting adults on each terminal, prior to spraying. Theplots were sprayed under reduced ambient light conditions and adultwhitefly numbers were assessed one day after treatment. Temperatureranged from 28-34° C. and relative humidity was 80-100%. Mean numbers ofwhiteflies/terminal and standard deviations were calculated for eachtreatment.

Results

Trial data are shown in FIG. 9. One day after treatment, there was nodetectable mortality in untreated, or PBO controls. Differences betweenwhitefly control and lambdacyhalothrin formulation were highlysignificant. Karate EC mixed with PBO provided some 25% control ofwhiteflies, while Karate Zeon+PBO gave virtually complete controlindicating complete suppression of resistance. Results are consistentwith the laboratory bioassay data.

Conclusions

Trial results indicate that very subdued daylight delayed the release oflambdacyhalothrin from encapsulation. There was a sufficient delaybetween the application of PBO and pyrethroid release frommicroencapsulation, to allow adequate inhibition of esterases by PBOprior to pyrethroid release. Therefore application of a synergist and adelayed release insecticide controlled highly pyrethroid resistantB-biotype B. tabaci in the field.

1-30. (canceled)
 31. A method for reducing resistance to a pesticide ofa substrate pest, comprising administering to a substrate or a pest: (a)a rapid-release formulation of a metabolic enzyme inhibitor; andsubstantially simultaneously, (b) a pesticide encapsulated in adegradable capsule, thereby reducing resistance of the substrate pest tothe pesticide.
 32. The method of claim 31, wherein component (a) andcomponent (b) are administered separately.
 33. The method of claim 32,wherein component (a) and component (b) are administered to thesubstrate or the pest within 10 seconds of each other.
 34. The method ofclaim 33, wherein component (a) and component (b) are administered tothe substrate or the pest within one or two seconds of each other. 35.The method of claim 31, wherein the rapid-release formulation ofcomponent (a) is a standard pesticide formulation, which comprises atleast one of the formulations selected from the group consisting of:wettable powders, granules, emulsifiable concentrates, and ultra-lowvolume formulations to which water can be added to form an emulsion or asuspension.
 36. The method of claim 31, wherein the inhibitor comprisesan esterase inhibitor.
 37. The method of claim 36, wherein the esteraseinhibitor comprises at least one compound selected from the groupconsisting of: S,S,S,-tributyl phosphorothionate; O,O,O-triphenylphosphate; piperonyl butoxide (PBO); profenofos; ethion; and dimethoate.38. The method of claim 31, wherein the inhibitor of component (a) ispiperonyl butoxide (PBO).
 39. The method of claim 31, wherein thecomposition comprises more than one pesticide encapsulated in adegradable capsule.
 40. The method of claim 39, wherein one or morepesticides are microencapsulated within a degradable capsule.
 41. Themethod of claim 31, wherein the pesticide of component (b) comprises atleast one compound selected from the group consisting of: pyrethroids;organo-phosphates; and carbamates.
 42. The method of claim 41, whereinthe pesticide of component (b) includes at least one pyrethroid selectedfrom the group consisting of: fenvalerate; S-fenvalerate; both alpha andzeta forms of cypermethrin; bifenthrin; deltamethrin; andbeta-cyfluthrin.
 43. The method of claim 31 wherein component (a) ispiperonyl butoxide and component (b) is a pyrethroid.
 44. A pesticidecomposition for reducing resistance to a pesticide of a substrate pestcomprising: (a) a rapid-release formulation of a metabolic enzymeinhibitor; and (b) a pesticide encapsulated in a degradable capsule. 45.The composition of claim 44, wherein component (a) and component (b) areformulated as an admixture.
 46. The composition of claim 44, wherein therapid-release formulation of component (a) is a standard pesticideformulation, which comprises at least one of the formulations selectedfrom the group consisting of: wettable powders, granules, emulsifiableconcentrates, and ultra-low volume formulations to which water can beadded to form an emulsion or a suspension.
 47. The composition of claim44, wherein the inhibitor of component (a) comprises at least oneinhibitor selected from the group consisting of: esterase inhibitors,mixed function oxidase inhibitors, and glutathione S-transferaseinhibitors.
 48. The composition of claim 47, wherein the inhibitor ofcomponent (a) is an esterase inhibitor.
 49. The composition of claim 44,wherein the pesticide of component (b) includes at least one compoundselected from the group consisting of: pyrethroids, organo-phosphates,and carbamates.
 50. The composition of claim 44, wherein the inhibitorof component (a) is piperonyl butoxide and the pesticide of component(b) is a pyrethroid.